JP5116473B2 - Cholesterol absorption inhibitor - Google Patents

Description

  The present invention relates to a Bifidobacterium bacterium excellent in cholesterol absorption suppression action, and a cholesterol absorption inhibitor containing these Bifidobacterium bacteria as active ingredients.

  Cholesterol is present in many cells and plays a major physiological role in maintaining cell functions as a component of lipoproteins and biological membranes, and as a raw material for bile acids and various hormones. ing. However, it is known that excessive intake of food with a high content and increased cholesterol in the serum may cause arteriosclerosis. It is required to suppress the concentration of

  And Bifidobacterium bifidum, Bifidobacterium addresscentis, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum cells have blood lipid improving action (Patent Document 1). In particular, research on Bifidobacterium longum has progressed, and it has been reported that Bifidobacterium longum SBT 2933R (FERM P-8743) has an excellent effect (Non-patent Document 1). It has been proposed to use this bacterial cell or culture as a serum cholesterol elevation inhibitor (Patent Document 2). However, in general, Bifidobacterium is weak against oxygen, gastric acid and bile acids in the body, so when ingesting these cells and cultures, the survival in the digestive tract is poor. There was a problem that sufficient effects were often not obtained. For this reason, a Bifidobacterium bacterium having an excellent inhibitory effect on survival of serum cholesterol is required.

  However, Bifidobacterium genus bacteria having excellent survival are known as Bifidobacterium longum SBT 10254 (FERM P-14820) (Patent Document 3) and Bifidobacterium Longum (FERM BP-7787) (patent document 4) has excellent survival and exhibits an inhibitory effect on serum cholesterol elevation, Bifidobacterium longum BB536, Bifidobacterium breve ATCC 15700, Bifidobacterium Animalis ATCC 25527 (Non-Patent Document 2) merely shows a cholesterol precipitation action, and there are few options for such microorganisms having excellent survival and cholesterol suppression action.

In addition, drugs and foods using these microorganisms are often required to be stored for a long period of time, so high storage stability is required, but conventionally known microorganisms have low survival after long-term storage. In particular, many of them have low survival after storage under non-anaerobic conditions.
Japanese Patent Application Laid-Open No. 61-271223 Japanese Patent Publication No. 6-96537 Japanese Patent No. 3384907 Japanese Patent Laid-Open No. 2003-238423 Proceedings of the 6th Annual Meeting of the Japan Bifidobacteria Center, p. 18, 1987 Letters in Applied Microbiology, Vol. 21, 149-151, 1995

  Therefore, the present invention is excellent in survival in the gastrointestinal tract, has an action to suppress absorption of cholesterol in the intestine, is excellent in lipid metabolism improving action such as lowering blood cholesterol, and has high survival after storage. It is an object of the present invention to provide a bacterium belonging to the genus Bacteria and a cholesterol absorption inhibitor using the same.

  As a result of intensive studies to achieve the above object, the present inventors have found that Bifidobacterium animalis subspices animalis YIT 10394, Bifidobacterium animalis subspices lactis JCM 1253, Bifidobacterium Animalis Subspices Lactis JCM 7117 and Bifidobacterium pseudolongum subspices globosum are surprisingly excellent in eliminating cholesterol, exhibiting strong acid resistance and bile acid resistance, and excellent in the intestine It was found that it has an absorption inhibitory action and has high survival after storage, and particularly shows high survival even after storage under non-anaerobic conditions. Among the microorganisms used in the present invention, Bifidobacterium animalis subsp. YIT 10393 is a microorganism newly found by the present inventors.

  That is, the present invention relates to Bifidobacterium animalis subspices animalis YIT 10394, Bifidobacterium animalis subspices lactis JCM 1253, Bifidobacterium animalis subspices lactis JCM 7117 and Bifidobacterium shoe The present invention provides a cholesterol absorption inhibitor comprising as an active ingredient one or more microorganisms selected from dronham, subspecies, and globosum.

  The present invention also provides a cholesterol absorption inhibitor comprising as an active ingredient one or more microorganisms selected from Bifidobacterium animalis subspices lactis JCM 1253 and Bifidobacterium animalis subspices lactis JCM 7117. Is.

  Further, the present invention is selected from Bifidobacterium animalis subspecies animalis YIT 10394, Bifidobacterium pseudolongum subspices globosum YIT 10392 and Bifidobacterium pseudolongum subspices globosum YIT 10393 The present invention provides a cholesterol absorption inhibitor comprising one or more microorganisms as active ingredients.

  Furthermore, the present invention relates to novel microorganisms such as Bifidobacterium animalis subspecies animals YIT 10394 (FERM ABP-10661), Bifidobacterium pseudolongum subspecies globosum YIT 10392 (FERM ABP-10660). Or Bifidobacterium pseudolongum subsp. Globosum YIT 10393 (FERM ABP-10661).

  Bifidobacterium animalis subspices animalis YIT 10394, Bifidobacterium animalis subspices lactis JCM 1253, Bifidobacterium animalis subspices lactis JCM 7117 and Bifidobacterium pseudolongum・ Subspecies globosum not only has an excellent cholesterol elimination effect, but also has excellent acid resistance and bile acid resistance, so it also exhibits excellent cholesterol absorption suppression action in the intestine, lowering blood cholesterol, etc. It can be used for the purpose of improving lipid metabolism. In addition, these microorganisms have high survival after storage, and in particular, show high survival even after storage under non-anaerobic conditions, the absorption inhibitor of the present invention can be stored for a long time, Non-anaerobic storage is also possible.

  Bifidobacterium pseudolongum subsp. Globosum used in the present invention includes, for example, Bifidobacterium pseudolongum subspices globosum YIT 10392, Bifidobacterium shoe Delongham Subspecies Globosum YIT 10393.

  These Bifidobacterium pseudolongum subspecies globosum YIT 10392, Bifidobacterium pseudolongum subspecies globosum YIT 10393, and Bifidobacterium animalis which is one of the microorganisms used in the present invention Bifidobacterium animalis subsp. Animalis YIT 10394 has a cholesterol elimination activity of 70% or more, a survival rate of 20% or more in an artificial gastric fluid, and a proliferation property in a bile acid-containing medium. Degree) and a novel strain isolated by the present inventors as a strain having a property of 100 or more. The strain has the following mycological properties. That is, the bacteriological properties of these bacteria are all Gram-positive polymorphic gonococci, non-spore-forming, non-motile, obligate anaerobic, can grow at 37 ° C, and are shown above. Bifidobacterium, pseudoron gum, subspices, globosum, Bifidobacterium, pseudoron gum, subspecies The properties of globosum and Bifidobacterium animalis subspices animalis were shown.

  Species identification for the novel strain was carried out by identifying the 16S rDNA base sequence of YIT 10392 in Bifidobacterium pseudolongum subspices globosum (accession No. D86194) and 99. .6% homology, 16S rDNA base sequence of YIT 10393 shows 99.6% homology with Bifidobacterium pseudolongum subsp. Globosum (accession No. D86194), and YIT It was based on the fact that 10394 16S rDNA base sequence showed 99.7% homology with Bifidobacterium animalis subsp. Animalis (accession No. D86185). Here, the bacterial species identification using the 16S rDNA base sequence was extracted from the bacterial cells that had been subjected to anaerobic (carbon dioxide substitution) culture at 37 ° C. for 24 hours using a GAM medium supplemented with 0.5% glucose and centrifuged. Using DNA as a template, the full length of the 16S rDNA sequence was amplified by the PCR method, the base sequence of the amplified product was determined by the Dye Terminator method, and the obtained base sequence was searched in the database. The judgment that these strains are novel was made on the basis that the 16S rDNA base sequence was different from that of the reference strain and that the results of chromosomal DNA polymorphism analysis by the RAPD (Random Amplified Polymorphic DNA) method were different.

  Bifidobacterium animalis subspecies animalis YIT 10394 is the microorganism accession number FERM ABP-10462, Bifidobacterium pseudolongum subspices globosum YIT 10392 is the microorganism accession number FERM ABP-10660, Bifidobacterium -Pseudolongum Subspecies Globosum YIT 10393 was transferred to the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology as of August 14, 2006, under the microorganism accession number FERM ABP-10661. The depositee before the transfer is the same, and the deposit date is August 18, 2005. The address of the deposit center is postal number 305-8656, 1st, 1st East, 1st Street, Tsukuba, Ibaraki, Japan.

  In addition, Bifidobacterium animalis subsp. Lactis JCM 1253 and Bifidobacterium animalis subsp. Lactis JCM 7117 used in the present invention are both Jol (Jiporis) and Col Jis 7 And ATCC (American Type Culture Collection), the ATCC numbers are ATCC 27536 and ATCC 27654, respectively.

  Among these, as the microorganism used in the present invention, the survival of the microorganism after producing a fermented milk food and drink using the microorganism and storing it under non-anaerobic conditions (aerobic conditions) at 10 ° C. for 21 days. Microorganisms with a rate of 50% or more are preferred.

  The microorganism of the present invention is preferably a Bifidobacterium bacterium having a cholesterol elimination activity of 70% or more, a cholesterol elimination activity of 70% or more, a survival rate of 20% or more in an artificial gastric juice, and a bile acid-containing microorganism. Bifidobacterium having a Klett value (bacterial turbidity) of 100 or more is more preferable. In general Bifidobacterium, the survival rate in artificial gastric juice is about 0 to several percent, and the growth in bile acid-containing medium is about 0 to several tens in terms of cret value (bacterial turbidity). . Bacteria with high acid resistance and bile acid resistance selected according to the above values can reach the intestine in a state in which many bacteria are alive.

Here, for the calculation of cholesterol elimination activity, for example, when microbial cells are allowed to stand together with artificial lipid micelles, the cholesterol in the supernatant is measured according to a conventional method, and compared with the amount of cholesterol in the supernatant not containing the cells. Therefore, it can be performed by applying the following equation.
Cholesterol elimination activity (%) = 100− (the amount of cholesterol in the supernatant containing cells) / (the amount of cholesterol in the supernatant not containing cells) × 100

  Moreover, the survival rate in artificial gastric juice can be made into the survival rate when preserve | saved at 37 degreeC and artificial gastric fluid of pH3.0 for 1 hour, for example. A high value indicates a high survival rate in the digestive tract.

  In addition, the growth in a bile acid-containing medium is, for example, the turbidity when cultured for 24 hours at 37 ° C. in a 0.2% bile acid-containing medium (Klettt-Summerson colorimeter, No. 66 filter). It can be as shown in A high value indicates that the survival rate and proliferation rate in the gastrointestinal tract are high.

  Note that the genus name, species name and strain name of microorganisms vary depending on the name of the person, and are also fluid for reclassification of bacteria, etc. However, microorganisms that are substantially the same as the microorganism are included in the microorganism of the present invention. That is, for example, Bifidobacterium animalis and Bifidobacterium lactis were sometimes treated as different species, but Masco et al. Summarized them as Bifidobacterium animalis and subclassified Bifidobacterium.・ Animals, Subspecies, Animalis and Bifidobacterium, Animalis, Subspecies, and Lactis have been reported as two subspecies (Int. J. Syst. Envol. Microbiol 54, 1137-1143, 2004). Therefore, the range of Bifidobacterium animalis subspices animalis or Bifidobacterium animalis subspices lactis of the present invention was previously referred to as Bifidobacterium animalis or Bifidobacterium lactis, respectively. The bacteria that had been included are included.

  Bifidobacterium animalis subsp. Lactis ATCC 27536 is the same as Bifidobacterium animalis subsp. Lactis JCM 1253, and Bifidobacterium animalis subsp. Lactis ATCC 27654 is Bifidobacterium. It is the same as Umm Animalis Subspecies Lactis JCM 7117 and is included in the microorganism of the present invention. In JCM and ATCC, both strains are classified as Bifidobacterium animalis, subspices, and animalis, but the 16S rDNA base sequences of these two strains are Bifidobacterium animalis subspices lactis (accession No. 2). X89513) and 100% coincidence, therefore, in the present invention, Bifidobacterium animalis subspices lactis was used.

  The microorganism of the present invention, as shown in the examples below, has cholesterol elimination activity and lowers cholesterol, particularly blood cholesterol concentration. It is useful as an inhibitor of cholesterol absorption from the intestinal tract. The cholesterol absorption inhibitor of the present invention is due to a decrease in blood cholesterol, a decrease in triglyceride, a decrease in VLDL and LDL cholesterol, a decrease in arteriosclerosis index, an increase in HDL cholesterol, and a postmenopausal female hormone deficiency. It is expected to be used to improve frequent hyperlipidemia and reduce the risk of developing arteriosclerosis.

  Moreover, since the microorganisms of this invention show acid tolerance and bile acid tolerance as shown in an Example below, what contains these effective amounts can be used as a cholesterol absorption inhibitor for oral administration.

  Furthermore, since the microorganisms of the present invention have high survival even when stored under aerobic conditions (non-anaerobic storage) in fermented milk foods and drinks, as shown in Examples below, those containing an effective amount thereof It can be used as a cholesterol absorption inhibitor that is stored under aerobic conditions (non-anaerobic storage). Storage temperature can be made into -80-10 degreeC, for example.

  Microorganisms included as an active ingredient in the cholesterol absorption inhibitor of the present invention include Bifidobacterium animalis, Subspecies, Animalis YIT 10394, Bifidobacterium animalis, Subspices, Lactis JCM 1253, Bifidobacterium animalis, Subspecies. One or more selected from Lactis JCM 7117 and Bifidobacterium pseudolongum subspices globosum may be used, but as a combination of microorganisms used here, for example, Bifidobacterium animalis subspices Lactis One or more combinations selected from JCM 1253 and Bifidobacterium animalis subsp. Lactis JCM 7117, Bifidobacterium animalis One or more combinations selected from Subspices Animalis YIT 10394, Bifidobacterium pseudolongum, Subspices globosum YIT 10392, and Bifidobacterium pseudolongum subspices globosum YIT 10393.

  In addition, the cholesterol absorption inhibitor of the present invention includes Bifidobacterium animalis subspices animalis YIT 10394, Bifidobacterium animalis subspices lactis JCM 1253, Bifidobacterium animalis subspices lactis JCM 7117 , And Bifidobacterium pseudolongum subspices globosum can be used alone or in combination, and other microorganisms can be used in combination. Examples of such microorganisms include Bifidobacterium animalis subspecies animals, Bifidobacterium animalis subspecies lactis, Bifidobacterium pseudolongum subspices globosum, Bifidobacterium addressensen Tis, Bifidobacterium angularum, Bifidobacterium asteroides, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenatum, Bifidobacterium denthium, Bifidobacterium Gallicum, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium pseudocatenatum, Bifidobacterium pseudo Bifidobacterium bacteria such as Ngam subspices pseudolongum, Bifidobacterium swiss, Bifidobacterium thermophilum; Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus casei , Lactobacillus john sony, Lactobacillus crispartus, Lactobacillus rhamnosus, Lactobacillus kefir, Lactobacillus delbruxi subspices delbrukii, Lactobacillus delbrucchi subspices bulgaricus, Lactobacillus helveticas, Lactobacillus sacrum , Lactobacillus reuteri, Lactobacillus Mali, Lactobacillus amylobolus, Lactobacillus amentariu , Lactobacillus bacteria such as Streptococcus thermophilus; Streptococcus bacteria such as Streptococcus thermophilus; Lactococcus bacteria such as Subspices cremolith, Lactococcus lactis subspices and Lactis; Leuconostoc genus bacteria such as Leuconostoc mesenteroides subspices cremolith; Pediococcus genus bacteria such as Pediococcus cerevisiae; Enterococcus bacteria such as Enterococcus faecalis; Acetobacter species such as Acetobacter aceti Bacteria; Gluconobacter bacteria such as Gluconobacter oxydans; Bacillus bacteria such as Bacillus subtilis; Saccharomyces yeasts such as Saccharomyces cerevisiae; Torlaspora yeasts such as Torlaspora delbruecki; Candida kefir Yeast of the genus Candida; yeast of the genus Kluyveromyces such as Krivellomyces marxianus; yeast of the genus Devariomyces such as Debaryomyces hansenii; yeast of the genus Pichia such as Pichia anomala; Aspergillus genus, Mucor genus, Monascus genus, Penici such as Mucor Japonicas, Monascus Purpleus, Penicillium Kamanberi, Rhizomucor psiras, Rhizopus alizas Umm spp., Rhizomucor spp., And the like molds of the genus Rhizopus.

  In addition, the microorganisms included in the cholesterol absorption inhibitor of the present invention may be freeze-dried, or can be used as a culture containing these microorganisms. It is preferable that it is a state of a living microbe.

  The cholesterol absorption inhibitor of the present invention can be administered in the form of a conventional pharmaceutical preparation by mixing the aforementioned microorganism with a solid or liquid nontoxic pharmaceutical carrier. Examples of such preparations include solid preparations such as tablets, granules, powders and capsules, liquid preparations such as solutions, suspensions and emulsions, and freeze-dried preparations. These preparations can be prepared by conventional means on the preparation. Examples of non-toxic pharmaceutical carriers include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, gelatin, albumin, water And physiological saline. Further, if necessary, conventional additives such as a stabilizer, a wetting agent, an emulsifier, a binder, and an isotonic agent can be appropriately added.

In addition, the cholesterol absorption inhibitor of the present invention can be ingested in the form of food or drink, in addition to ingesting the microorganism as it is or by directly adding it to food. Preferred examples of the food and drink include fermented milk, fermented soymilk, fermented fruit juice, fermented vegetable liquid, fermented rice juice, fermented wort and fermented food and drink of animal and plant-derived materials containing the microorganisms of the present invention in the state of viable bacteria. (Pickles, miso, soy sauce, fermented tea, fermented sausage, fermented mayonnaise, cheese, salted salt, etc.) can be mentioned, and these foods and drinks can be produced by ordinary methods. For example, in the case of producing fermented milk, first, Bifidobacterium animalis subspices animalis YIT 10394, Bifidobacterium animalis subspices lactis JCM 1253, Bifidobacterium animalis subspices are put into a sterilized milk medium. At least one or more of Lactis JCM 7117 and Bifidobacterium pseudolongum subspices globosum are inoculated and cultured alone or simultaneously with other microorganisms and homogenized to obtain a fermented milk base. Then, a separately prepared syrup solution is added and mixed, homogenized with a homogenizer or the like, and further added with flavor to finish the final product. The fermented milk of the present invention thus obtained can be made into a product of any form such as a plain type, a soft type, a fruit flavor type, a solid form, a liquid form, and a frozen form. The food and drink includes animal feed. The microorganism concentration of the present invention in the fermented milk can be, for example, 10 ³ to 10 ¹³ cfu / ml.

  In this case, the cholesterol absorption inhibitor of the present invention includes food materials used in ordinary foods and drinks, for example, sugars, proteins, peptides, lipids, vitamins, minerals, vegetable components such as vegetables, grains, fruits, blood, Animal components such as milk, liver, bones, muscles, microbial components such as bacteria, mold, yeast, mushrooms or culture components, gelling agents, solidifying agents, thickeners, fragrances, coloring agents, Bifidobacterium bacteria A growth promoter, a lactic acid bacteria growth promoter, etc. can be mix | blended. Specifically, glucose, sucrose, fructose, maltose, glucose fructose liquid sugar, various sweeteners such as xylose, palatinose, honey, maple syrup, amazake, sorbitol, xylitol, erythritol, lactitol, palatinit, reduced starch syrup, reduced maltose Various sugar alcohols such as syrup, various high sweetness sweeteners such as sucralose and aspartame, various natural sweeteners such as licorice, stevia and glycyrrhizic acid glycosides, various emulsifiers such as sucrose fatty acid ester, glycerin sugar fatty acid ester and lecithin And various thickening (stabilizing) agents such as agar, gelatin, carrageenan, guar gum, gum arabic, xanthan gum, pectin, locust bean gum and the like.

  In addition, tagatose, lactose, trehalose, trehalose, agarooligosaccharide, nigerooligosaccharide, galactooligosaccharide, fructooligosaccharide, xylooligosaccharide, raffinose, stachyose, lactulose, maltotriose, isomaltoligosaccharide, cyclodextrin, glucosamine, N-acetyl Various carbohydrates such as glucosamine, alginic acid, sodium alginate, fucoidan, salgassan, flucelan, funolan, porphyran, aminaran, pullulan, tara gum, konjac mannan, inulin, chitin, chitosan, polydextrose, hyaluronic acid, chondroitin sulfate, β-glucan, Mannan, galactan, fructan, xylan, arabinan, arabinogalactan, glucomannan, galactomannan, beet Fiber, oat fiber, wheat fiber, soybean fiber, rice fiber, barley fiber, xanthan gum, corn fiber, apple fiber, citrus fiber, silum fiber, pine fiber, prune fiber, pea fiber, banana fiber, acetic acid bacteria bacterial cellulose, Lactobacillus cell walls, Bifidobacterium cell walls, yeast cell walls, natto fructan, collagen, natto polyglutamic acid and other dietary fibers, hydrolysates of these dietary fibers, wheat bran, barley bran, rice Bran, bran bran, oat bran, rye bran, rye bran, cyrus, rice bran, brown rice, chicory, soybean okara, apple pulp, resistant starch, barley malt, corn seed outside Skin, Lactic acid bacteria, Bifidobacterium bacteria, Beer yeast, Wine yeast, Wine koji, Sake lees, Soy sauce koji, Beer koji, Rice koji, Wheat koji, Bean koji, Red koji, Yellow koji Natto mucilage, grape seed extract, royal jelly, propolis, chlorella, spirulina, euglena, wakame, kombu, hondawala, arame, kajime, asakusanori, aonori, hijiki, aosa, mozuku, etc. Various materials are listed.

  Furthermore, various minerals such as calcium, magnesium, zinc, iron, manganese, iodine, selenium, copper, cobalt, dolomite or various salts thereof, citric acid, malic acid, tartaric acid, pyruvic acid, gluconic acid, succinic acid, Various acids such as fumaric acid, ascorbic acid, lactic acid, acetic acid, propionic acid, butyric acid, phosphoric acid, creatine, methionine, cysteine, glutamic acid, etc. or various salts of these acids, glutathione, phytin, phytic acid, lignin, poly -Γ-glutamic acid and its degradation products, saponin, ferulic acid, γ-aminobutyric acid, γ-oryzanol, chalcone, flavanone, flavone, flavonol, isoflavone, anthocyan, catechin, proanthocyanidins, tea polyphenol, curcumid, capsai Oids, sesaminol, sesameignan, theaflavins, β diketones, carotenoids, allyl sulfur compounds, isothiocyanates, terpenes, chlorophylls, sphingolipids, gangliosides, n-3 polyunsaturated fatty acids, n-6 polyvalent Various components such as unsaturated fatty acids, conjugated linoleic acids, phospholipids, plant sterols, soybean proteins such as glycinin and conglycinin, egg proteins such as ovalbumin and ovomucoid, milk proteins and milk such as casein, lactalbumin, and lactoferrin Protein, rice protein such as casein phosphopeptide, oryzenin, wheat protein such as glutenin and gliadin, various proteins such as fish protein, and their enzymatic and acid-degraded peptides, vitamin A, vitamin B group, vitamin C, Vitamin D group, Vitamin E, Vitamin K group, various vitamins such as β-carotene, retinoic acid, folic acid, black cohosh, pumpkin seeds, pomegranate seeds, hypericum, passionflower, valerian, puelleria mirifica, rosemary , Peppermint, parsley, marigold, lemon balm, mugwort, safflower, radish seeds, coffee tree, sorghum, sorghum fruit, citrus pericarp, ginkgo biloba, jujube, kokushi, licorice, ganoderma, ginseng, guarana And various plant extracts such as green tea, black tea, oolong tea, gymnema tea and guava leaves, and various spices such as pepper, salamander, cinnamon, turmeric, sage, thyme, basil, pepper, and nutmeg.

  In addition, various cereal components such as rice, brown rice, barley, wheat, oats, rye, pearl barley, amaranth, millet, millet, buckwheat, sorghum, corn, etc., or various germinated components of seeds of these cereals, azuki bean, white azuki bean, quince bean , Kidney beans, peas, purple beans, black beans, black soybeans, green soybeans, mung bean, broad beans, dairy bears, ashitaba, kale, turmeric, potatoes, sweet potatoes, purple sweet potatoes, yams, pumpkins, eggplants, tomatoes, bitter melons, cabbages, cabbages , Broccoli, cauliflower, lettuce, green beans, ginger, burdock, celery, Japanese radish, horseradish, avocado, carrot, spinach, onion, garlic, lily, octopus, perilla, leek, leek, parsnip, bracken, bamboo shoot Various vegetable ingredients such as shiitake mushroom, lemon, apple, grape, strawberry, orange, oyster, guava, banana, blueberry, blackberry, cranberry, raspberry, cowberry, yam, feijoa, tamarillo, acerola, lime, seeker, melon, Peach, mango, yuzu, papaya, pineapple, pear, plum, grapefruit, karin, apricot, mandarin orange, pomegranate, watermelon, prunes, kiwi and other fruit ingredients, peanut, almond, coconut, cashew nut, macadamia nut, cacao, chestnut, ginnan Nuts, such as walnuts, milk, skim milk, whey, cream, fermented milk, yogurt, milk protein, casein, whey protein, and other dairy products and their components, sake, grape wine, Shaoxing liquor, beer Brewing liquor, whiskey, brandy, and a distillation liquor like vodka like.

  For fermented milk foods and beverages using Bifidobacterium, containers made of oxygen-impermeable packaging materials such as glass and aluminum-coated paper are mainly used for the purpose of enhancing the survival during storage. However, as shown in the examples below, the microorganism of the present invention has high oxygen resistance and does not require a strict anaerobic state. Therefore, the container material of the cholesterol absorption inhibitor of the present invention has oxygen permeability. High resin (polystyrene, polyethylene, polyethylene terephthalate, etc.) can also be used. Containers using these resins have merits such as low cost and high degree of freedom of molding compared to containers made of oxygen-impermeable packaging.

  Since the microorganism which is an active ingredient of the cholesterol absorption inhibitor of the present invention exhibits excellent acid resistance as shown in the examples below, the cholesterol absorption inhibitor of the present invention can be made acidic, for example, 25 The pH at ° C. can be 2-7, especially 3-6.

Since the Bifidobacterium genus bacterium, which is an active ingredient of the cholesterol absorption inhibitor of the present invention, has been conventionally used as food and its safety has been confirmed, it is used as a cholesterol absorption inhibitor. Although there is no strict restriction | limiting in the dosage in the case, The suitable dosage is 10 < ⁵ > cfu-10 < ¹³ > cfu per day as a viable count, and especially 10 < ⁸ > cfu-10 < ¹² > cfu is preferable.

EXAMPLES Hereinafter, although an Example and a test example are given and this invention is demonstrated in detail, this invention is not restrict | limited at all by these.
[Test Example 1]
(1) Cholesterol elimination activity The culture solution anaerobically cultured at 37 ° C. for 24 hours in m-ILS medium (Int. J. Food Microbiol. 81.131-136 2003) was centrifuged at 12,000 rpm for 15 minutes at 4 ° C. The cells were washed with a 150 mM phosphate buffer having a pH of 5.5. Centrifugal washing was repeated three times, and then suspended in 150 mM phosphate buffer so that the turbidity (absorbance at 660 nm) was 3 (10 ⁸ to 10 ⁹ cfu / ml as the number of bacteria). Prepared. The cell suspension was autoclaved at 121 ° C. for 15 minutes to prepare a dead cell suspension.
2 g of beef bile powder, 921 mg of cholesterol, 135 mg of lysophosphatidylcholine, 90.2 mg of monooleic acid and 702.2 mg of oleic acid were suspended in 150 mM phosphate buffer (pH 7.0) and sonicated for 12 minutes. Thereafter, ultracentrifugation was performed at 100,000 G for 18 hours at 25 ° C., and the micelle layer was collected to prepare artificial lipid micelles. 150 μl of artificial lipid micelle was added to 1 ml of the bacterial cell suspension (10 ⁸ to 10 ⁹ cfu as the number of bacteria), and the mixture was allowed to stand at 37 ° C. After 18 hours, cholesterol in the supernatant centrifuged at 12,000 rpm for 15 minutes at 4 ° C. was measured with Detamina TC555 (Kyowa Medix). As a control, a phosphate buffer solution containing no bacterial cells was subjected to the same operation, and cholesterol elimination activity was calculated according to the following formula. Further, the cholesterol elimination activity was similarly calculated for the dead cell suspension.
Cholesterol elimination activity (%) = 100− (the amount of cholesterol in the supernatant containing cells) / (the amount of cholesterol in the supernatant not containing cells) × 100

(2) Acid resistance To 10 ml of 50 mM Na ₂ HPO ₄ solution adjusted to pH 3 with hydrochloric acid, 0.1 ml of the bacterial solution cultured to the stationary phase in GAM medium (Nissui) was added, treated at 37 ° C. for 1 hour, Survival rate was calculated.
Survival rate (%) = (viable cell count after acid treatment) / (viable cell count before acid treatment) × 100

(3) Bile acid resistance 30 μl of the bacterial solution cultured in a GAM medium until stationary phase was inoculated into 3 ml of GAM medium supplemented with 0.2% bile acid and cultured anaerobically at 37 ° C. After 24 hours, microbial turbidity was measured with a Klett-Summerson colorimeter (No. 66 filter).

  The test results of the above (1) to (3) are shown in Table 1.

  As shown in Table 1, Bifidobacterium animalis subspecies animals, Bifidobacterium animalis subspecies lactis and Bifidobacterium pseudolongum subspecies globosum of the present invention have high cholesterol elimination activity. It was excellent in acid resistance and bile acid resistance. Moreover, since dead cells did not have cholesterol elimination activity, it was found that these bacteria showed activity in the state of live bacteria.

  Next, when the cholesterol in the precipitate was measured for the purpose of knowing whether the disappearance of cholesterol from the supernatant was due to precipitation or due to conversion or decomposition, the cholesterol lost from the supernatant was not converted or decomposed. It was revealed that it was present in the precipitate. From the above, it was suggested that the cholesterol in the supernatant was taken up and precipitated in the cells, or the lipid micelles were disintegrated and precipitated due to the deconjugation action of bile acids of the cells. .

[Test Example 2] Effects on blood lipids and liver lipids of animals 10% non-fat dry milk medium containing 0.03% yeast extract was sterilized at 121 ° C for 15 minutes, and Bifidobacterium pseudolongum subspices 1% globosum YIT 10393 was inoculated and cultured anaerobically for 24 hours. This culture solution was inoculated 2% in the same medium and cultured anaerobically at 37 ° C. for 46 to 54 hours. The number of viable bacteria contained in the fermented milk thus prepared was 3.7 × 10 ⁸ cfu / ml. This fermented milk was freeze-dried to prepare a feed having the composition shown in Table 2. Here, the viable cell count in the bait was 1.0 × 10 ⁶ cfu / g. On the other hand, as a control, a 10% skim milk medium containing 0.03% yeast extract was sterilized at 121 ° C. for 15 minutes, and then freeze-dried without inoculation with bacteria as unfermented milk. The composition shown in Table 2 Bait was prepared.

  Next, Wistar strain ovariectomized rats (Japan SLC) or sham-operated rats purchased at 8 weeks of age were acclimated to AIN-93G purified diet for 1 week after preliminary breeding for 1 week, and shown in Table 3 according to body weight. They were grouped into experimental groups.

The rats thus grouped were reared on the test diet for 34 days in individual bracket cages at room temperature 24 ± 1 ° C. and humidity 55 ± 5 ° C. under conditions allowing free intake of food and water corresponding to each group. . Daily viable count intake was about 10 ⁷ cfu / animal.
Next, the feeder was removed, and 4 hours later, Nembutal anesthesia was performed, and abdominal aortic blood was collected and the liver was collected by perfusion. The liver was stored at −20 ° C. until analysis, and the blood was centrifuged at 3000 rpm for 15 minutes to separate the plasma. The liver was lyophilized and then extracted by the method of Folch (J. Biol. Chem. 226, 497-509, 1957).
The amounts of total cholesterol and triglyceride contained in the plasma and liver extract thus obtained were determined using Detamina TC555 (Kyowa Medix) and Triglyceride E Test Wako (Wako Pure Chemical), respectively. Moreover, the amount of HDL cholesterol contained in plasma was determined using HDL-cholesterol E test Wako (Wako Pure Chemical Industries). Furthermore, the amount of VLDL + LDL-cholesterol contained in plasma was determined by subtracting the amount of HDL cholesterol from the total amount of cholesterol.
Based on the obtained results, a two-way analysis of variance was performed on the ovariectomized group to determine p. In addition, a t-test was performed on the ovariectomized group administered with food similar to the sham operation group. The obtained results are shown in Tables 4-6. Values were expressed as mean ± SD. In Tables 4 to 6, the interaction indicates an interaction between fermented milk and galactooligosaccharide.

表４に示すとおり、卵巣摘出ラットは偽手術群と比較して、既報通り（Ｊ．Ｃｏｍｐ．Ｐｈｙｓｉｏｌ．Ｐｈｙｓｉｃｏｌ ８８：１８３−１９３、１９７５）体重や摂食量が多くなったが、卵巣摘出群の中ではオリゴ糖の有無や発酵乳投与の影響は認められず、良好に成長した。

As shown in Table 4, the ovariectomized rats had increased body weight and food intake as reported (J. Comp. Physiol. Physicol 88: 183-193, 1975), compared with the sham operation group. Among them, the presence or absence of oligosaccharides or the influence of fermented milk administration was not observed, and the cells grew well.

  Moreover, as shown in Table 5, the effect of fermented milk prepared with Bifidobacterium pseudolongum subspices globosum YIT 10393 is recognized in blood cholesterol, triglyceride, VLDL + LDL-cholesterol, arteriosclerosis index, The item was also reduced. In particular, the decrease in VLDL + LDL-cholesterol was significant. In addition, the effects of oligosaccharides on these items and the interaction between fermented milk and oligosaccharide were not observed statistically, but total blood cholesterol, triglyceride, VLDL + LDL− in the combined administration group of oligosaccharide and fermented milk were not observed. The values of cholesterol and arteriosclerosis index were the lowest. It is known that galactooligosaccharides are specifically used by Bifidobacterium. Therefore, it was shown that by administering fermented milk containing galactooligosaccharide, Bifidobacterium, pseudoron gum, subspices, globosum YIT 10393 grows in the intestine, and effectively reduces blood lipids. In addition, it is known that by removing the ovaries, blood total cholesterol increases, and it is useful as a postmenopausal hyperlipidemia model. Comparison of data between the oophorectomy / unfermented milk / no oligosaccharide group and the sham operation group showed that the oophorectomy was successful in this study.

As shown in Table 6, the presence or absence of oligosaccharides or the presence or absence of administration of fermented milk had no effect on the liver cholesterol content, and there was no lipid accumulation in the liver. In the case of triglyceride, no statistically significant difference was observed per liver by administration of fermented milk, but the triglyceride content per gram of liver decreased.
As described above, fermented milk prepared with Bifidobacterium pseudolongum subspices globosum YIT 10393 has reduced total blood cholesterol, triglyceride, VLDL + LDL-cholesterol, arteriosclerosis index, and cholesterol only. However, it was revealed that it has the ability to improve lipid metabolism for various blood lipids and has the effect of reducing the risk of developing arteriosclerosis.

[Test Example 3] Examination of survival in fermented milk 0.03% yeast extract was added to 10% nonfat milk and sterilized, and the bacteria of the present invention (Bifidobacterium animalis, subspices animalis) YIT 10394, Bifidobacterium animalis subspices lactis JCM 1253, JCM 7117, Bifidobacterium pseudolongum subspices globosum YIT 10392, YIT 10393), each at a temperature of 37 ° C. By culturing to 1 ± 1.0, five types of fermented milk were produced. Moreover, fermented milk was similarly produced using Bifidobacterium longum ATCC 15707 as a control.
The prepared fermented milk was dispensed into glass test tubes in the case of anaerobic storage, and 10 ml each in polypropylene tubes in the case of non-anaerobic storage (aerobic storage), and stored at 10 ° C. for 12 weeks. In the case of anaerobic preservation, it was sealed with a butyl rubber stopper under a N ₂ stream, and in the case of non-anaerobic preservation, the polypropylene tube lid was loosely tightened. Table 7 shows the pH and the number of viable bacteria at the end of the culture, Table 8 shows the transition of the number of bacteria under non-anaerobic storage, and Table 9 shows the transition of the number of bacteria under anaerobic storage.

As shown in Table 8, in non-anaerobic conditions (aerobic conditions) storage, Bifidobacterium longum ATCC 15707 used as a control decreased to 9.2 × 10 ³ cfu / ml after 3 weeks, and 8 weeks It was not detected afterwards. In contrast, the Bifidobacterium bacterium of the present invention does not fall below 1 × 10 ⁸ cfu / ml for any strain stored for 3 weeks, and the survival rate when compared to the end of the culture is It became more than 50%. In addition, Bifidobacterium animalis subsp. Lactis JCM 1253 and JCM 7117 do not fall below 1 × 10 ⁸ cfu / ml even after storage for 12 weeks, and the remaining 3 strains (Bifidobacterium pseudolongum・ Subspecies Globosum YIT 10392, YIT 10393, Bifidobacterium animalis, Subspecies animalis YIT 10394) also decreased by about 1 to 2 orders.
As shown in Table 9, in anaerobic storage, the number of Bifidobacterium longum ATCC 15707 decreased to about 1/10 after storage for 3 weeks. The bacterial count at the time was almost maintained. Bifidobacterium pseudolongum subspices globosum YIT 10392, YIT 10393, Bifidobacterium animalis subspices animalis YIT 10394 is only about 1/2 to 1/6 even if stored for 12 weeks The number of Bifidobacterium animalis subsp. Lactis JCM 1253 and JCM 7117 was almost the same after 12 weeks.

[Prescription Example 1] Manufacture of tablets Various ingredients were mixed according to the following formulation, granulated, dried and sized, and then tableted to produce tablets.
(Prescription) (mg)
Dried product of bacterial cells of the present invention ¹⁾ 20
Microcrystalline cellulose 100
Lactose 80
Magnesium stearate 0.5
Methylcellulose 12
¹⁾ : Bifidobacterium pseudolongum subsp. Globosum YIT10392 live cells were obtained by freeze-drying.

[Prescription Example 2] Manufacture of fermented milk beverage Sterilized by adding 3% glucose to 15% skimmed milk, inoculated with 1% Bifidobacterium pseudolongum subspices globosum YIT 10393, and at 35 ° C for 24 hours Anaerobic culture was performed to obtain 210 g of a fermented milk base. On the other hand, 97 g of sugar and 0.2 g of emulsified iron were dissolved in water and sterilized with a total amount of 790 g to obtain a syrup. The fermented milk base and syrup obtained as described above were mixed, and 1 g of flavor was added, and then homogenized at 15 MPa and filled in a container to obtain a fermented milk drink. The obtained fermented milk beverage had good appearance and flavor, and the viable cell count immediately after production was 2.5 × 10 ⁸ cfu / ml. The viable cell count after storage at 10 ° C. for 21 days was 1.4 × 10 ⁸ cfu / ml, and the storage stability was also good.

[Prescription Example 3] Production of Soft Drink According to the following prescription, each component was blended and homogenized according to a conventional method to obtain a soft drink. The obtained soft drink was filled in a brown bottle, sealed with an aluminum cap, and heat-treated. The resulting soft drink had good appearance and flavor and good storage stability.
(Prescription) (g)
Dried product of bacterial cells of the present invention ¹⁾ 5
Fragrance 0.8
Water 100
Reduced starch saccharified product 24
Fructose 18
¹⁾ : Bifidobacterium animalis subsp. Animalis YIT 10394 live cells were freeze-dried to obtain.

Claims (2)

  One or more microorganisms selected from Bifidobacterium animalis subspices animalis YIT 10394, Bifidobacterium pseudolongum subspices globosum YIT 10392 and Bifidobacterium pseudolongum subspices globosum YIT 10393 Cholesterol absorption inhibitor as an active ingredient.   Bifidobacterium animalis subspecies animalis YIT 10394 (FERM ABP-10661), Bifidobacterium pseudolongum subspices globosum YIT 10392 (FERM ABP-10660), or Bifidobacterium pseudoron gum Subspecies Globosum YIT 10393 (FERM ABP-10661).